Fatigue is the decline in force or power when muscles are used intensively. The mechanisms have been studied in isolated single muscle fibres which are amenable to intracellular measurements. However single fibres lack the restriction of O2 supply and the accumulation of K+ and lactic acid which occur in intact preparations. In an attempt to avoid these deficiencies we studied the intact tibialis anterior muscles of anaesthetised mice (112 mg/kg ketamine + 1.5 mg/kg medetomidine i.p.) with intact blood supply and the distal tendon attached to a force transducer. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100 Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5 % (SE, n=13)of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4 % after 4 min. Intracellular phosphate concentration ([Pi]) was measured by 31P nuclear magnetic resonance on perchloric acid extracts of muscles. [Pi] increased during fatigue from 7.6 ± 1.7 (SE, n=6) to 16.0 ± 1.6 mmol/kg muscle wet weight and returned to control during recovery. Intracellular calcium was measured with cameleons whose plasmids had been transfected in the muscle 2 weeks before the experiment. Yellow cameleon 2 was used to measure myoplasmic calcium and D1ER to measure sarcoplasmic reticulum (SR) calcium. The myoplasmic calcium during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR calcium also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [Pi] and a reduction in the tetanic myoplasmic calcium. We suggest that both changes contribute to the observed fatigue. A likely cause of the decline in tetanic myoplasmic calcium is precipitation of calcium phosphate in the SR.